title: Characterization of an in vitro differentiation assay for pancreatic-like cell development from murine embryonic stem cells: detailed gene expression analysis.
abstract: Embryonic stem (ES) cell technology may serve as a platform for the discovery of drugs to treat diseases such as diabetes. However, because of difficulties in establishing reliable ES cell differentiation methods and in creating cost-effective plating conditions for the high-throughput format, screening for molecules that regulate pancreatic beta cells and their immediate progenitors has been limited. A relatively simple and inexpensive differentiation protocol that allows efficient generation of insulin-expressing cells from murine ES cells was previously established in our laboratories. In this report, this system is characterized in greater detail to map developmental cell stages for future screening experiments. Our results show that sequential activation of multiple gene markers for undifferentiated ES cells, epiblast, definitive endoderm, foregut, and pancreatic lineages was found to follow the sequence of events that mimics pancreatic ontogeny. Cells that expressed enhanced green fluorescent protein, driven by pancreatic and duodenal homeobox 1 or insulin 1 promoter, correctly expressed known beta cell lineage markers. Overexpression of Sox17, an endoderm fate-determining transcription factor, at a very early stage of differentiation (days 2-3) enhanced pancreatic gene expression. Overexpression of neurogenin3, an endocrine progenitor cell marker, induced glucagon expression at stages when pancreatic and duodenal homeobox 1 message was present (days 10-16). Forced expression (between days 16 and 25) of MafA, a pancreatic maturation factor, resulted in enhanced expression of insulin genes, glucose transporter 2 and glucokinase, and glucose-responsive insulin secretion. Day 20 cells implanted in vivo resulted in pancreatic-like cells. Together, our differentiation assay recapitulates the proceedings and behaviors of pancreatic development and will be valuable for future screening of beta cell effectors.
authors.affiliation: Department of Diabetes, Endocrinology, and Metabolism, Beckman Research Institute of City of Hope, Duarte, California, USA.
authors.forename: Chialin
authors.initials: C
authors.lastname: Chen
authors.forename: Jing
authors.initials: J
authors.lastname: Chai
authors.forename: Lipi
authors.initials: L
authors.lastname: Singh
authors.forename: Ching-Ying
authors.initials: CY
authors.lastname: Kuo
authors.forename: Liang
authors.initials: L
authors.lastname: Jin
authors.forename: Tao
authors.initials: T
authors.lastname: Feng
authors.forename: Scott
authors.initials: S
authors.lastname: Marzano
authors.forename: Sheetal
authors.initials: S
authors.lastname: Galeni
authors.forename: Nan
authors.initials: N
authors.lastname: Zhang
authors.forename: Michelina
authors.initials: M
authors.lastname: Iacovino
authors.forename: Lihui
authors.initials: L
authors.lastname: Qin
authors.forename: Manami
authors.initials: M
authors.lastname: Hara
authors.forename: Roland
authors.initials: R
authors.lastname: Stein
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Bromberg
authors.forename: Michael
authors.initials: M
authors.lastname: Kyba
authors.forename: Hsun Teresa
authors.initials: HT
authors.lastname: Ku
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D019070:Cell Lineage / Q000235:genetics; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology / Q000502:physiology*; D004707:Endoderm / Q000166:cytology / Q000378:metabolism; D051526:Fibroblast Growth Factor 10 / Q000096:biosynthesis / Q000378:metabolism; D015870:Gene Expression; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D008297:Male; D051379:Mice; D016513:Mice, SCID; D010179:Pancreas / Q000166:cytology / Q000254:growth & development* / Q000378:metabolism; D055760:SOXF Transcription Factors / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D015534:Trans-Activators / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
chemical_list: C496463:FGF10 protein, human; D051526:Fibroblast Growth Factor 10; D018398:Homeodomain Proteins; D007328:Insulin; C109188:SOX17 protein, human; D055760:SOXF Transcription Factors; D015534:Trans-Activators; D014157:Transcription Factors; C485184:enhanced green fluorescent protein; C084010:pancreatic and duodenal homeobox 1 protein; D049452:Green Fluorescent Proteins